Products of proteolytic cleavage of transferrin induce nitric oxide response of goldfish macrophages

Enzymatic cleavage product of transferrin induced the production of nitric oxide (NO) by LPS-stimulated goldfish macrophages. A NO-inducing factor was purified from the supernatants of mitogen-stimulated goldfish kidney leuko cytes using fast performance liquid chromatography (FPLC) and the purified proteins analyzed by microcapillary reverse-phase HPLC nanoelectrospray tan dem mass spectrometry. The proteins were identified as truncated forms of t ransferrin, having approximate molecular weights (MW) of 33, 35, and 37 kDa (kilodaltons). The precursor form (i.e. full-length) of transferrin did no t enhance NO production by LPS-stimulated goldfish macrophages, but enzymat ic cleavage of this precursor form correlated with enhanced production of N O by goldfish macrophages. Enzymatic cleavage of transferrin was dependent on the presence of stimulated kidney leukocytes and was shown to occur in r esponse to both mixed lymphocyte reactions (MLR) and the mitogenic stimulat ion of goldfish kidney leukocytes. Time course analysis revealed that 24 h after kidney leukocyte MLR or mitogen stimulation, cleaved transferrin prod ucts appeared in the supernatants of cultured cells, which was related to t he on-set of NO-inducing activity of these preparations. To confirm these f indings, bovine transferrin was digested in vitro using protease XXVII. The resulting cleavage products had approximate MW of 33, 35, and 37 kDa. When these peptides were subjected to the purification protocols used to purify a NO-inducing factor from goldfish leukocyte supernatants, they were shown to elute to identical fractions. To examine the potential role of fish tra nsferrin in mediating goldfish NO production, carp transferrin was purified from serum and following protease-digestion and purification by FPLC, the truncated proteins were found to elute to similar fractions as bovine trans ferrin, Furthermore, mitogen-stimulated leukocyte supernatants prepared in the absence of bovine serum (carp serum only) retained NO-inducing activity , indicating that this response was not an artifact of bovine serum compone nts (i.e. bovine transferrin), Anti-bovine and anti-carp transferrin polycl onal antibodies identified the presence of truncated forms of transferrin i n the active fractions of FPLC-separated mitogen-stimulated leukocyte super natants prepared in the presence of bovine or carp serum, respectively. Thu s, our results suggest a novel role for fish transferrin as one of the fact ors that mediates teleost macrophage antimicrobial functions. (C) 2000 Else vier Science Ltd. All rights reserved.